Results from the research proposed here should illuminate much of the molecular logic of how Cys and Ser residues in a ribosomally synthesized peptide are processed by microcin B17 synthetase to a potent antibiotic containing four thiazole and four oxazole rings. Intermediates in this multi-step process, trapped at several stages by time course studies, will be structurally characterized by Electrospray Ionization/Fourier- transform Mass Spectrometry (FTMS) without prior separation or degradation in solution. This analysis should yield insight into the regioselectivity and processivity of the synthetase for the eight modification sites. Structural analogs of a truncated substrate, such as alpha-CH3- alkylation and isotopic labeling, combined with the FTMS- based assay should allow assessment of the substrate tolerance and stereoselectivity of the synthetase, and force some intermediates of ring formation to accumulate to test an initial mechanistic proposal involving cyclization, dehydration, and dehydrogenation. Photoaffinity labeling should tag residues near the ATP binding site, with identification of these residues indicating candidates for site-directed mutagenesis to knock out ATP binding. Detailed mechanisms for the unique chemistry of this antibiotic biogenesis could facilitate future development of antibiotic compounds to replace or complement those to which some bacteria have evolved a resistance.